Astaxanthin anti-inflammatory synergistic combinations

ABSTRACT

Compositions having synergistic combinations of astaxanthin with a tomato extract lycopene, and optionally with carnosic acid and/or lutein. Compositions having synergistic combinations of the aforementioned compounds, which may be used, inter alia, to inhibit/suppress inflammation via the suppression of the expression of anti-inflammatory mediators or via the suppression of the secretion of anti-inflammatory mediators from macrophages at a site of inflammation.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/778,800 filedSep. 21, 2015, which is a U.S. national phase of InternationalApplication No. PCT/IL2014/050272 filed Mar. 13, 2014, which claims thebenefit of the filing date of U.S. Provisional Patent Application Ser.No. 61/803,191, filed Mar. 19, 2013. The contents of the aboveapplications are all incorporated by reference as if fully set forthherein in their entirety.

FIELD OF INVENTION

This invention is directed to; inter alia, compositions comprisingsynergistic combinations of astaxanthin with lycopene, and optionallywith carnosic acid and/or lutein. More specifically, the presentinvention provides a composition comprising a synergistic combination ofthe aforementioned compounds, which may be used, inter alia, toinhibit/suppress inflammation.

BACKGROUND OF THE INVENTION

The inflammatory process, which forms an important part of thenon-specific immune system, is characterized by a complex set ofchemical and cellular changes that are essential for host defense in theface of microbial agents and other potentially harmful environmentalfactors. However, in many cases, inflammation may be triggeredinappropriately, and/or may persist to a degree which becomes harmful tothe host. In such cases, there may be a need to inhibit or prevent thedevelopment of one or more aspects of the inflammatory process, inparticular, in cases of non-infectious inflammatory diseases.

A very large number of different chemical mediators have been shown tobe involved in the development and control of the inflammatory process.Recent studies by a number of different laboratories have implicatednitric oxide (NO) as an important modulator of a variety of acute andchronic inflammatory disorders, including various types of arthritis,gastro-intestinal diseases, inflammatory conditions of the centralnervous system and certain forms of asthma. Consequently, it has beenproposed that inhibition of NO production could provide a usefultherapeutic mechanism for the treatment and/or management of theseinflammatory disorders. Furthermore, inhibition of NO synthesis has alsobeen shown to be useful in some conditions or states that are notprimarily inflammatory in nature. Thus, for example, inhibition of NOsynthesis has been found to reduce glucose uptake into limb tissue inindividuals with Type 2 diabetes during exercise.

The in vivo production of NO is mediated by a family of nitric oxidesynthase (NOS) enzymes, including inducible-nitric oxide synthase(I-NOS), which is activated by many different immunological stimuliincluding lipopolysaccharide (LPS), interferon gamma and interleukin 1(IL-1).

Several other compounds, including a number of natural products, havealso been shown to inhibit NO production. The latter group includescompounds such as lutein [Rafi M. M. et al. Mol Nutr Food Res. 2007March; 51(3):333-40; Choi, J. S. Nutrition. 2006 June; 22(6):668-71] andlycopene [Rafi, M. M. et al. J Food Sci. 2007 January; 72(1):S069-74].However, the efficacy and potency of many of the natural product NOinhibitors have proven to be not particularly high. A need thereforeexists for improved NO production-inhibiting compositions of naturalorigin.

Another highly important inflammatory mediator is the tumor necrosisfactor-alpha (TNF-alpha), which is a cytokine produced by a variety ofcell types including macrophages, neutrophils and lymphocytes. TNF-alphaoccupies a key position in the early stage of the inflammatory processand is responsible for stimulating the production of other factors suchas nuclear factor-κB which in turn causes activation of a wide range ofpro-inflammatory genes. Thus, in view of its key pro-inflammatory role,TNF-alpha is clearly an important potential therapeutic target foranti-inflammatory agents.

It is a purpose of the present invention to provide a composition thatmay be used to inhibit the production of one or more key inflammatorymediators, such as superoxide NO or TNF-alpha, as a means for treatingor managing pathological states and processes in which said mediatorsare implicated.

It is another purpose of the invention to provide a composition that isable to inhibit the production of the aforesaid inflammatory mediatorswith greater efficacy and/or potency than the compounds and compositionsreported in the prior art.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising tomato lycopene such as but not limited to Lyc-O-Mato® andastaxanthin.

In another embodiment, the present invention further provides acomposition comprising astaxanthin, carnosic acid, and tomato lycopene.

In another embodiment, the present invention further provides acomposition comprising astaxanthin, lutein, and tomato lycopene.

In another embodiment, the present invention further provides acomposition comprising astaxanthin, carnosic acid, lutein, and tomatolycopene.

In another embodiment, the present invention further provides that thecomposition further comprises lutein, phytoene, phytofluene,beta-carotene, tocopherols, phytosterols, or any combination thereof. Inanother embodiment, the present invention further provides that themolar concentration ratio of astaxanthin to tomato lycopene is from 6:1to 1:2. In another embodiment, the present invention further providesthat the molar concentration ratio of astaxanthin to carnosic acid isfrom 1:1 to 1:10. In another embodiment, the present invention furtherprovides that the molar concentration ratio of astaxanthin to tomatolycopene to carnosic acid is from 1:2:1 to 1:5:10. In anotherembodiment, the present invention further provides that the compositionhas a synergistic anti-inflammatory effect.

In another embodiment, the present invention further provides a methodfor treating a subject afflicted with inflammation, comprising the stepof administering to the subject a therapeutically effective amount of acomposition comprising: (1) astaxanthin, (2) lutein and/or carnosicacid, and (3) tomato lycopene, thereby treating a subject afflicted withinflammation. In another embodiment, the present invention furtherprovides that treating a subject afflicted with inflammation isinhibiting the production of NO, PGE2, TNF-alpha, or any combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are bar graphs showing the effect of various compositions ofthe invention including combinations of active ingredients on the NOproduction by LPS-stimulated macrophages. FIG. 1A provides a compositionwhich includes astaxanthin (AST); FIG. 1B provides a composition whichincludes AST and carnosic acid (CA); FIG. 1C provides a compositionwhich includes tomato lycopene extract and AST; and FIG. 1D provides acomposition which includes lutein and AST. The effect of thesecompositions on the production of NO in LPS induced macrophages wastested. The Y axis provides a measure of inhibition of NO production byLPS-stimulated macrophages.

FIGS. 2A-2C Are bar graphs showing the effect of various compositions ofthe invention (including combinations of active ingredients) on the NOproduction by LPS-stimulated macrophages: FIG. 2A is AST, tomatolycopene extract, and carnosic acid (CA); AST, FIG. 2B is tomatolycopene extract, and lutein; and FIG. 2C is AST, tomato lycopeneextract, lutein, and carnosic acid on the production of NO in LPSinduced macrophages.

FIGS. 3A-3D are bar graphs showing the effect of various compositions ofthe invention (including combinations of active ingredients) on theTNF-alpha production by LPS-stimulated macrophages: FIG. 3A isastaxanthin (AST); FIG. 3B is AST and carnosic acid (CA); FIG. 3C istomato lycopene extract and AST; and FIG. 3D is lutein and AST on theproduction of TNF-alpha in LPS induced macrophages.

FIGS. 4A-4C are bar graphs showing the effect of various compositions ofthe invention (including combinations of active ingredients) on theTNF-alpha production by LPS-stimulated macrophages: FIG. 4A is AST,tomato lycopene extract, and carnosic acid (CA); FIG. 4B is AST, tomatolycopene extract, and lutein; and FIG. 4C is AST, tomato lycopeneextract, lutein, and carnosic acid on the production of TNF-alpha in LPSinduced macrophages.

FIG. 5 is a bar graph showing the anti-inflammatory effect (NOproduction of LPS induced macrophages) of synthetic lycopene versustomato extract lycopene.

FIGS. 6A-6B are bar graphs representing superoxide production by nonstimulated peritoneal cells. The cells were activated (FIG. 6B) orprimed (FIG. 6A) due to the thioglicollate peritoneal injection andreflect the situation of the cells in a site of inflammation. As shownthere is a significant (p<0.01) reduction (39%) in the release ofsuperoxide by the nutrient mixture treatment compared with the placebo(3.82+1.52 compared with 6.22±1.53 nmoles O₂/10⁶ cells/min,respectively).

FIGS. 7A-7C are bar graphs showing the reduction (significance p<0.05)in NO production (FIG. 7A) by peritoneal cells harvested from miceundergoing nutrient drinking compared with placebo; 2.35+0.36 comparedwith 3.72+0.88 μM, respectively. Similarly, there is a reduction(significant p<0.05) in PGE₂ production (FIG. 7B) and TNF alphaproduction (FIG. 7C) by peritoneal cells harvested from mice undergoingthe nutrient drink of the invention compared with placebo, 1.75+0.26compared with 2.72+0.33 ng/ml.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising astaxanthin and tomato lycopene. In one embodiment, thepresent invention provides a composition comprising astaxanthin,carnosic acid, and tomato lycopene. In one embodiment, the presentinvention provides a composition comprising astaxanthin, lutein, andtomato lycopene. In one embodiment, the present invention provides acomposition comprising astaxanthin, lutein, carnosic acid, and tomatolycopene.

In another embodiment, tomato lycopene is lycopene extracted from atomato plant. In another embodiment, tomato lycopene is a tomato extractenriched for lycopene. In another embodiment, tomato lycopene is alycopene-rich tomato extract which is all-natural. In anotherembodiment, tomato lycopene is a tomato lycopene complex. In anotherembodiment, tomato lycopene complex comprises a complex ofphytonutrients including phytoene, phytofluene, beta-carotene,tocopherols and phytosterols. In another embodiment, tomato lycopene isLyc-O-Mato® (LycoRed Ltd., Be'er Sheva, Israel). In another embodiment,a composition of the invention comprises Lyc-O-Mato® and astaxanthin.

Suitable processes for preparing this extract and similar extracts aredescribed in U.S. Pat. No. 5,837,311, the specification of which isincorporated herein by reference in its entirety. However, it is to berecognized that many other types of preparatory procedures may be usedto obtain the composition from a variety of plant sources.

In another embodiment, asthaxanthin is an ester of mainly di-palmitate.In another embodiment, astaxanthin is extracted and/or purified frommicroalgae. In another embodiment, astaxanthin is extracted and/orpurified from yeast. In another embodiment, astaxanthin is extractedand/or purified from salmon. In another embodiment, astaxanthin isextracted and/or purified from trout. In another embodiment, astaxanthinis extracted and/or purified from krill. In another embodiment,astaxanthin is extracted and/or purified from shrimp. In anotherembodiment, astaxanthin is extracted and/or purified from crayfish. Inanother embodiment, astaxanthin is extracted and/or purified from acrustacean. In another embodiment, astaxanthin is extracted and/orpurified from a feathers bird. In another embodiment, astaxanthin issynthetic astaxanthin. In another embodiment, synthetic astaxanthin isproduced by a synthesis path such as but not limited to the synthesispath described in Krause, Wolfgang; Henrich, Klaus; Paust, Joachim; etal. Preparation of Astaxanthin. DE 19509955.9 Mar. 18, 1995.

In another embodiment, astaxanthin is produced in bacteria via anengineered production system. In another embodiment, astaxanthinbiosynthesis proceeds from beta-carotene via either zeaxanthin orcanthaxanthin. In another embodiment, astaxanthin is the 3R,3′Rstereoisomer, the 3R,3′S (meso) stereoisomer, the 3S,3′S stereoisomer,or any combination thereof. In another embodiment, astaxanthin containsa mixture of the three stereoisomers in approximately 1:2:1 proportion.

In another embodiment, astaxanthin, which is a naturally-occurringcompound and is a potent antioxidant, is used in a composition of theinvention for ameliorating and retarding, and/or preventing,inflammatory or inflammatory induced traumatic disease or injury. Inanother embodiment, the composition as described herein has asynergistic ability to act as an anti-inflammatory, antioxidant.

In another embodiment, a composition as described herein furthercomprises phytoene. In another embodiment, a composition as describedherein further comprises phytofluene. In another embodiment, acomposition as described herein further comprises beta-carotene. Inanother embodiment, a composition as described herein further comprisestocopherol. In another embodiment, a composition as described hereinfurther comprises a phytosterol. In another embodiment, a composition asdescribed herein further comprises a combination of any two or more of:phytoene, phytofluene, beta-carotene, tocopherol, and phytosterol. Inanother embodiment, phytoene, phytofluene, beta-carotene, tocopherol,and phytosterol are of natural source. In another embodiment, phytoene,phytofluene, beta-carotene, tocopherol, and phytosterol are derived fromtomato. In another embodiment, phytoene, phytofluene, beta-carotene,tocopherol, phytosterol, or any combination thereof is producedsynthetically. In another embodiment, phytosterol can be a combinationof phytosterols.

In another embodiment, the molar concentration ratio of astaxanthin totomato lycopene is from 20:1 to 1:5. In another embodiment, the molarconcentration ratio of astaxanthin to tomato lycopene is from 15:1 to1:2. In another embodiment, the molar concentration ratio of astaxanthinto tomato lycopene is from 10:1 to 1:5. In another embodiment, the molarconcentration ratio of astaxanthin to tomato lycopene is from 6:1 to1:5. In another embodiment, the molar concentration ratio of astaxanthinto tomato lycopene is from 6:1 to 1:2.

In another embodiment, a composition as described comprises from 2.5 to15 mg tomato lycopene. In another embodiment, a composition as describedcomprises 5 mg tomato lycopene. In another embodiment, a composition asdescribed comprises from 5 to 10 mg tomato lycopene. In anotherembodiment, a composition as described comprises 0.5 to 100 mgastaxanthin. In another embodiment, a composition as described comprises10 to 100 mg astaxanthin. In another embodiment, a composition asdescribed comprises 10 to 50 mg astaxanthin. In another embodiment, acomposition as described comprises 20 to 50 mg astaxanthin. In anotherembodiment, a composition as described comprises 10 mg to 10 g carnosicacid. In another embodiment, a composition as described comprises 10 mgto 1 g carnosic acid. In another embodiment, a composition as describedcomprises 10 to 500 mg carnosic acid. In another embodiment, acomposition as described comprises 50 to 500 mg carnosic acid. Inanother embodiment, a composition as described comprises 100 to 500 mgcarnosic acid.

In another embodiment, the molar concentration ratio of astaxanthin tocarnosic acid in a composition of the invention is from 1:1 to 1:50. Inanother embodiment, the molar concentration ratio of astaxanthin tocarnosic acid in a composition of the invention is from 1:1 to 1:20. Inanother embodiment, the molar concentration ratio of astaxanthin tocarnosic acid in a composition of the invention is from 1:1 to 1:10. Inanother embodiment, the molar concentration ratio of astaxanthin tocarnosic acid in a composition of the invention is from 1:1 to 1:5.

In another embodiment, the molar concentration ratio of astaxanthin totomato lycopene to carnosic acid in a composition of the invention isfrom 1:1:1 to 1:20:40. In another embodiment, the molar concentrationratio of astaxanthin to tomato lycopene to carnosic acid in acomposition of the invention is from 1:1:1 to 1:10:20. In anotherembodiment, the molar concentration ratio of astaxanthin to tomatolycopene to carnosic acid in a composition of the invention is from1:1:1 to 1:10:10. In another embodiment, the molar concentration ratioof astaxanthin to tomato lycopene to carnosic acid in a composition ofthe invention is from 1:2:1 to 1:5:10.

In another embodiment, a composition of the invention further compriseszeaxanthin. In another embodiment, a composition of the inventionfurther comprises lutein. In another embodiment, lutein comprises(3R,3′R,6′R)-beta, epsilon-carotene-3,3′-diol. In another embodiment,lutein is a plant lutein. In another embodiment, lutein is tomatolutein. In another embodiment, lutein is marigold lutein. In anotherembodiment, lutein is provided as a marigold extract. In anotherembodiment, lutein is a synthetic lutein. In another embodiment, luteincomprises saponifying fatty acid.

The components of the above-disclosed compositions may be purifiedcompounds, synthetic compounds or may be present in mixture with othercomponents, for example in plant extracts such as rosemary extract (inthe case of carnosic acid), marigold extract (in the case of lutein) ora tomato extract (such as Lyc-O-Mato® which is commercially availablefrom LycoRed, Be'er Sheva, Israel—in the case of lycopene and othercarotenoids). In some embodiments, carnosic acid is supplied as rosemaryextract. In some embodiments, carnosic acid is obtained from a rosemaryextract.

In some embodiments, a composition as described herein has a synergisticanti-inflammatory effect. In some embodiments, a composition asdescribed herein is an oral composition. In some embodiments, acomposition as described herein further comprises a pharmaceutical or anutraceutical acceptable excipient.

In some embodiments, a composition as described herein inhibits theproduction and/or secretion of inflammatory mediators and cytokineswhich play important roles in the pathogenesis of a vast number ofmammalian inflammatory diseases. In another embodiment, a composition ofthe invention is a phytonutrient combination causing an immediate,efficient, and synergistic inhibition of LPS-induced internal superoxideproduction leading to a marked decrease in ERK and NF-kB activation.

In another embodiment, the present invention further provides a methodfor treating a subject afflicted with inflammation or septic shock,comprising the step of administering to the subject a therapeuticallyeffective amount of a composition as described herein. In anotherembodiment, the present invention further provides a method forinhibiting the production of proinflammatory cytokines, such as but notlimited to TNF-alpha by macrophages and monocytes at inflammatory sitescomprising the step of administering to a subject in need thereof, atherapeutically effective amount of a composition as described herein.In another embodiment, the present invention further provides a methodfor inhibiting the release of proinflammatory cytokines, such as but notlimited to TNF-alpha by macrophages and monocytes at inflammatory sitescomprising the step of administering to a subject in need thereof, atherapeutically effective amount of a composition as described herein.

In another embodiment, the present invention further provides a methodfor treating a subject afflicted with inflammation, wherein theinflammation is an inherent part of a disease state. In anotherembodiment, the present invention further provides a method for treatingrheumatoid arthritis. In another embodiment, the present inventionfurther provides a method for treating Crohn's disease. In anotherembodiment, the present invention further provides a method for treatingulcerative colitis. In another embodiment, the present invention furtherprovides a method for treating septic shock syndrome. In anotherembodiment, the present invention further provides a method for treatingatherosclerosis. In another embodiment, the present invention furtherprovides a method for treating juvenile rheumatoid arthritis. In anotherembodiment, the present invention further provides a method for treatingpsoriatic arthritis. In another embodiment, the present inventionfurther provides a method for treating osteoarthritis. In anotherembodiment, the present invention further provides a method for treatingrefractory rheumatoid arthritis. In another embodiment, the presentinvention further provides a method for treating chronic non-rheumatoidarthritis. In another embodiment, the present invention further providesa method for treating osteoporosis/bone resorption. In anotherembodiment, the present invention further provides a method for treatingendotoxic shock. In another embodiment, the present invention furtherprovides a method for treating ischemia-reperfusion injury. In anotherembodiment, the present invention further provides a method for treatingcoronary heart disease. In another embodiment, the present inventionfurther provides a method for treating vasculitis. In anotherembodiment, the present invention further provides a method for treatingamyloidosis. In another embodiment, the present invention furtherprovides a method for treating multiple sclerosis. In anotherembodiment, the present invention further provides a method for treatingsepsis. In another embodiment, the present invention further provides amethod for treating chronic recurrent uveitis. In another embodiment,the present invention further provides a method for treating hepatitis Cvirus infection. In another embodiment, the present invention furtherprovides a method for treating malaria. In another embodiment, thepresent invention further provides a method for treating ulcerativecolitis. In another embodiment, the present invention further provides amethod for treating cachexia. In another embodiment, the presentinvention further provides a method for treating plasmocytoma. Inanother embodiment, the present invention further provides a method fortreating endometriosis. In another embodiment, the present inventionfurther provides a method for treating Behcet's disease. In anotherembodiment, the present invention further provides a method for treatingWegenrer's granulomatosis. In another embodiment, the present inventionfurther provides a method for treating an autoimmune disease. In anotherembodiment, the present invention further provides a method for treatingankylosing spondylitis. In another embodiment, the present inventionfurther provides a method for treating common variable immunodeficiency(CVID). In another embodiment, the present invention further provides amethod for treating chronic graft-versus-host disease. In anotherembodiment, the present invention further provides a method for treatingtrauma and transplant rejection. In another embodiment, the presentinvention further provides a method for treating adult respiratorydistress syndrome. In another embodiment, the present invention furtherprovides a method for treating pulmonary fibrosis. In anotherembodiment, the present invention further provides a method for treatingrecurrent ovarian cancer. In another embodiment, the present inventionfurther provides a method for treating a lymphoproliferative disease. Inanother embodiment, the present invention further provides a method fortreating refractory multiple myeloma. In another embodiment, the presentinvention further provides a method for treating myeloproliferativedisorder. In another embodiment, the present invention further providesa method for treating diabetes. In another embodiment, the presentinvention further provides a method for treating juvenile diabetes. Inanother embodiment, the present invention further provides a method fortreating meningitis. In another embodiment, the present inventionfurther provides a method for treating skin delayed typehypersensitivity disorders. In another embodiment, the present inventionfurther provides a method for treating Alzheimer's disease. In anotherembodiment, the present invention further provides a method for treatingsystemic lupus erythematosus. In another embodiment, the presentinvention further provides a method for treating any other clinicalcondition which is inherently associated or depends on an inflammatoryprocess.

In another embodiment, the present invention provides that treating asubject afflicted with inflammation is inhibiting the production of ananti-inflammatory cytokine, a glucocorticoid, an anti-inflammatoryneuropeptide, or a lipid inflammation mediator. In another embodiment,the present invention provides that treating a subject afflicted withinflammation is inhibiting the production of NO, PGE, TNF-alpha, or anycombination thereof at a site of inflammation. In another embodiment,the present invention provides that treating a subject afflicted withinflammation is inhibiting the production of NO, PGE, TNF-alpha, or anycombination thereof by macrophages. In another embodiment, the presentinvention provides that treating a subject afflicted with inflammationis inhibiting the recruitment of neutrophils to the site ofinflammation. In another embodiment, the present invention provides thattreating a subject afflicted with inflammation is inhibiting neutrophilsactivation at the site of inflammation. In another embodiment, PGE isPGE2 (prostaglandin E2).

Furthermore, the present invention also provides a method of treatmentof pathological conditions in which superoxide ions, NO, TNF-alphaand/or PGE2 act as a modulator or mediator of the condition in amammalian subject in need of such treatment, wherein the methodcomprises administering to the subject a therapeutic compositionaccording to any one of the embodiments disclosed hereinabove.

In another aspect, the present invention is directed to the use of acomposition such as described herein for the manufacture of a medicamentfor the treatment of conditions responsive to inhibition of NO,TNF-alpha and/or PGE2 production.

In some embodiments of the methods described hereinabove, the subject isa human subject. In some embodiments of the methods describedhereinabove, the subject is a mammal. In some embodiments of the methodsdescribed hereinabove, the subject is a pet. In some embodiments of themethods described hereinabove, the subject is a farm animal. In someembodiments of the methods described hereinabove, the subject is a labanimal.

While in the above-disclosed methods, the therapeutic composition may beadministered by any convenient means, in one embodiment the compositionis administered in a pharmaceutical, a nutraceutical, nutritional, ororal dosage form. In another preferred embodiment, however, thetherapeutic composition is incorporated into a foodstuff or beverage.

In one embodiment, the composition of the present invention can beprovided to the individual per-se. In one embodiment, the composition ofthe present invention can be provided to the individual as part of afurther pharmaceutical composition or a nutraceutical composition whereit is mixed with a pharmaceutically acceptable carrier.

In one embodiment, a “pharmaceutical composition” or a “nutraceuticalcomposition” refers to a preparation of a composition as describedherein with other chemical components such as physiologically suitablecarriers and excipients. The purpose of a pharmaceutical composition ora nutraceutical composition is to facilitate administration of thecomposition to an organism.

In one embodiment, “a combined preparation” defines especially a “kit ofparts” in the sense that the combination partners as defined above canbe dosed independently or by use of different fixed combinations withdistinguished amounts of the combination partners i.e., simultaneously,concurrently, separately or sequentially. In some embodiments, the partsof the kit of parts can then, e.g., be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Theratio of the total amounts of the combination partners, in someembodiments, can be administered in the combined preparation. In oneembodiment, the combined preparation can be varied, e.g., in order tocope with the needs of a patient subpopulation to be treated or theneeds of the single patient which different needs can be due to aparticular disease, severity of a disease, age, sex, or body weight ascan be readily made by a person skilled in the art.

In one embodiment, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to a mammal and does not abrogate the biological activity andproperties of the administered composition. An adjuvant is includedunder these phrases.

In one embodiment, “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference in itsentirety.

In one embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

In one embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body. In one embodiment,the region of a patient's body is characterized by inflammation or ascomprising inflammatory mediators.

Various embodiments of dosage ranges are contemplated by this invention.The dosage of the composition of the present invention, in oneembodiment, is in the range of 0.5-2000 mg/day. In another embodiment,the dosage is in the range of 5-500 mg/day. In another embodiment, thedosage is in the range of 500-2000 mg/day. In another embodiment, thedosage is in the range of 0.1-10 mg/day. In another embodiment, thedosage is in the range of 50-500 mg/day. In another embodiment, thedosage is in the range of 5-4000 mg/day. In another embodiment, thedosage is in the range of 0.5-50 mg/day. In another embodiment, thedosage is in the range of 5-80 mg/day. In another embodiment, the dosageis in the range of 100-1000 mg/day. In another embodiment, the dosage isin the range of 1000-2000 mg/day. In another embodiment, the dosage isin the range of 200-600 mg/day. In another embodiment, the dosage is inthe range of 400-1500 mg/day. In another embodiment, the dosage is in arange of 800-1500 mg/day. In another embodiment, the dosage is in therange of 500-2500 mg/day. In another embodiment, the dosage is in arange of 600-1200 mg/day. In another embodiment, the dosage is in therange of 1200-2400 mg/day. In another embodiment, the dosage is in therange of 40-60 mg/day. In another embodiment, the dosage is in a rangeof 2400-4000 mg/day. In another embodiment, the dosage is in a range of450-1500 mg/day. In another embodiment, the dosage is in the range of1500-2500 mg/day. In another embodiment, the dosage is in the range of5-10 mg/day. In another embodiment, the dosage is in the range of550-1500 mg/day. In another embodiment, “dosage” refers to the amount ofan active ingredient or the combination of active ingredients of theinvention. In another embodiment, “dosage” is not inclusive with respectto excipients. Aqueous solutions, buffers, vehicles, or any other inertsubstance.

In one embodiment, the dosage is 200 mg/day. In another embodiment, thedosage is 300 mg/day. In another embodiment, the dosage is 400 mg/day.In another embodiment, the dosage is 500 mg/day. In another embodiment,the dosage is 600 mg/day. In another embodiment, the dosage is 700mg/day. In another embodiment, the dosage is 800 mg/day. In anotherembodiment, the dosage is 900 mg/day. In another embodiment, the dosageis 1000 mg/day.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,drinks, syrups, nectars, beverages, emulsions and the like. Such unitdosage forms comprise a safe and effective amount of the composition.The pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmellose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the active ingredient asknown to one skilled in the art. In another embodiment, the compositionis a drink or a beverage comprising a dosage which consists acombination of the active ingredients in a ratio or in an amount asdescribed herein.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like. In some embodiments,pharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. In some embodiments, liquid oralcompositions comprise from about 0.012% to about 0.933% of the activeingredients, or in another embodiment, from about 0.033% to about 0.7%.

In some embodiments, pharmaceutical compositions for use in the methodsof this invention comprise solutions or emulsions, which in someembodiments are aqueous solutions or emulsions comprising a safe andeffective amount of the composition of the present invention andoptionally, other compounds. In some embodiments, the compositionscomprise from about 0.01% to about 10.0% w/v or w/w of a combination ofactive ingredients as described herein.

Further, in another embodiment, the compositions are administeredtopically to body surfaces, and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the composition of the present invention are combinedwith an additional appropriate therapeutic agent or agents, prepared andapplied as solutions, suspensions, or emulsions in a physiologicallyacceptable diluent with or without a pharmaceutical carrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, compositions for use in accordance with the presentinvention is formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. In one embodiment,formulation is dependent upon the route of administration chosen.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In some embodiments, compositions include aqueous solutions of theactive preparation in water-soluble form. Additionally, suspensions ofthe active ingredients, in some embodiments, are prepared as appropriateoily or water based suspensions. Suitable lipophilic solvents orvehicles include, in some embodiments, fatty oils such as sesame oil, orsynthetic fatty acid esters such as ethyl oleate, triglycerides orliposomes. Aqueous suspensions contain, in some embodiments, substances,which increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol or dextran. In another embodiment, thesuspension also contain suitable stabilizers or agents which increasethe solubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

In some embodiments, compositions suitable for use in context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose. Insome embodiments, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

Some examples of substances which can serve as nutraceutical orpharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of a nutraceutical ora pharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the nutraceutical or pharmaceutically-acceptable carrier issterile, physiological saline, with a blood-compatible suspending agent,the pH of which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material, thecompositions of the invention, into or onto particulate preparations ofpolymeric compounds such as polylactic acid, polglycolic acid,hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellaror multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance. Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g. poloxamers or poloxamines).

In some embodiments, preparation of effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the compositiondescribed herein can be determined by standard nutraceutical orpharmaceutical procedures in vitro, in cell cultures or experimentalanimals. In one embodiment, the data obtained from these in vitro andcell culture assays and animal studies can be used in formulating arange of dosage for use in human. In one embodiment, the dosages varydepending upon the dosage form employed and the route of administrationutilized. In one embodiment, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. [See e.g., Fingl, et al., (1975) “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical or nutraceuticalcarrier are also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the composition. In oneembodiment, the pack, for example, comprise metal or plastic foil, suchas a blister pack. In one embodiment, the pack or dispenser device isaccompanied by instructions for administration. In one embodiment, thepack or dispenser is accommodated by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of nutraceuticals or pharmaceuticals, whichnotice is reflective of approval by the agency of the form of thecompositions or human or veterinary administration. Such notice, in oneembodiment, is labeling approved by the U.S. Food and DrugAdministration for prescription drugs or of an approved product insert.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include chemical, molecular,biochemical, and cell biology techniques. Such techniques are thoroughlyexplained in the literature. See, for example, “Molecular Cloning: Alaboratory Manual” Sambrook et al., (1989); “Current Protocols inMolecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); “CellBiology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed.(1994); The Organic Chemistry of Biological Pathways by John McMurry andTadhg Begley (Roberts and Company, 2005); Organic Chemistry ofEnzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002);Organic Chemistry (6th Edition) by Leroy “Skip” G Wade; OrganicChemistry by T. W. Graham Solomons and, Craig Fryhle.

Material and Methods

Cells

Macrophage isolation and culture Peritoneal macrophages were collectedfrom the peritoneal cavity of 6- to 8-week-old male ICR mice (Harlan,Israel) after an intraperitoneal injection of 1.5 ml ofthioglycollatebroth (4%) 4 days before harvest. Peritoneal macrophageswere washed three times with phosphate-buffered saline (PBS) and, whenappropriate, a hypotonic lysis of erythrocytes was performed, yielding ahighly enriched (90-95%) macrophage cell population. Macrophages wereidentified by FACS (Becton-Dickinson, Mountain View, Calif., USA)analysis using FITC-conjugated rat anti-mouse F4/80 (MCA497F; Serotec,Oxford, UK) by flow microfluorimetry. For each sample, 10,000light-scatter-gated viable cells were analyzed. Peritoneal macrophages(1×10⁶ cells/well) were cultured in 96-well-plates at 37° C. in a 5% CO₂atmosphere in RPMI 1640 medium containing 10% fetal calf serum, 2 mML-glutamine. 100 U/ml penicillin. 100 mg/ml streptomycin (Beit-Haemek,Israel). Cells were stimulated with 1 mg/ml LPS from Salmonella entericaserotype typhimurium in the absence or presence of different combinationas described herein (see the figures in the examples section). Thephytonutrients were dissolved in dimethylsulfoxide (DMSO; in a finalconcentration of 5 mM). The mixture was vortexed and incubated in awater bath at 37° C. (with shaking) for 10 mins and sonicated in asonicator bath for 15 seconds three times. Working concentrations of thecompounds were prepared from the stock solution by addition ofappropriate volumes to warmed culture medium. The final concentration insolution was calculated by addition of 0.5 ml isopropanol and 1.5 mlhexane/dichloromethane (1/5 v/v) containing 0.025% BHT to 1 ml of theculture medium. The solution was vortexed and the liquid phases wereseparated by centrifugation at 3000 rpm for 10 min. Spectrophotometrywas performed to measure the concentrations of lycopene, asthaxanthin,lutein, b-carotene. To the controls appropriate volumes of DMSO(0.1-0.2%) were added and the percentage inhibition in each tube testwas calculated in relation to its control.

TNF-Alpha Production Assay

Concentrations of TNF-alpha in cell culture supernatants were quantifiedby ELISA kits (Biolegend, SanDiego, Calif., USA).

Example 1 The Anti-Inflammatory Effect of the Composition of the PresentInvention on Macrophages

The anti-inflammatory effect was detected by inhibition of NO productionand of TNF alpha production by LPS-stimulated macrophages.

The figures below show that astaxanthin inhibited NO (FIG. 1) and TNFalpha production (FIG. 3) in a dose dependent manner.

This experiment proved the synergetic inhibition (p<0.001 compared withthe additive effect) by combinations of astaxanthin in the range of0.5-2 μM with 1 μM Tomato Lycopene extract LycoMato (LM), but not with 1μM lutein or 1 μM carnosic acid (FIGS. 1 and 3). The letter S above thehorizontal lines determine the synergistic effect

Addition of 1 μM carnosic acid to the combinations of astaxanthin in therange of 0.1-1 μM with 1 μM LycoMato (LM) caused a synergetic inhibition(p<0.001 compared with the additive effect) (FIGS. 2 and 4) that washigher than the effect achieved in the absence of carnosic acid.

The inhibitory effect of the combinations of astaxanthin with LycoMatoand lutein was lower than that of astaxanthin with LycoMato and carnosicacid (FIGS. 2 and 4).

Finally, the effect of the four phyto-nutrients was not significantlydifferent from the effect of asthaxanthin with LycoMato and carnosicacid (FIGS. 2 and 4).

Example 2: Tomato Lycopene is Far More Effective than Synthetic Lycopenein its Anti-Inflammation Activity as Measured by NO Production

Tomato lycopene extract was found to be far more effective thansynthesis lycopene in reducing NO production by macrophages (FIG. 5).Synergistic inhibition for the secretion of proinflammatory mediators byLPS-stimulated macrophages pre-incubated with combinations ofLyc-O-Mato® with lutein, astaxanthin, b-carotene, and carnosic acid, wasrecorded. This novel combination was found to be unexpectedlyefficacious for the current medical indications.

Moreover, a synergistic inhibition for the production of NO byLPS-stimulated macrophages pre-incubated with a combination ofLyc-O-Mato® (LM) and astaxanthin was recorded (FIG. 1).

As shown in FIG. 2 a synergistic inhibition for the production of NO byLPS-stimulated macrophages pre-incubated with a combination of: (1)Lyc-O-Mato® (LM), carnosic acid, and astaxanthin or (2) Lyc-O-Mato®(LM), lutein, and astaxanthin, were recorded.

As shown in FIG. 3 a synergistic inhibition for the production ofTNF-alpha by LPS-stimulated macrophages pre-incubated with a combinationof Lyc-O-Mato® (LM) and astaxanthin was recorded.

As shown in FIG. 3 a synergistic inhibition for the production ofTNF-alpha by LPS-stimulated macrophages pre-incubated with a combinationof: (1) Lyc-O-Mato® (LM), carnosic acid, and astaxanthin, (2)Lyc-O-Mato® (LM), lutein, and astaxanthin, or (3) Lyc-O-Mato® (LM),carnosic acid, lutein and astaxanthin, were recorded.

Example 3: The In-Vivo Anti-Inflammatory Effect of a Mixture TomatoLycopene Extract+Astaxanthin+Carnosic Acid was Determine in a MouseModel of Sterile Peritonitis

Methods

Mouse Model of Sterile Peritonitis—

Six to eight weeks old male ICR mice (Harlan Laboratories, Israel),average weight 30 gr, were fed ad libitum rodent chow (#19520 Kofolk,Pethach Tikva, Israel) and free reverse osmosis filtered water. Animalswere housed in static microisolator cages with sterile pine shavingbedding in 12:12 light dark cycles 18-26 Celsius degrees and 30-70%relative humidity. Mice received in their drinking water asupplementation of Lyc-O-Mato®, Astaxanthin, Carnosic acid at the ratioof 1:0.5:1 (The quantity of Lyc-O-Mato® used was 10 mg/kg) during 7 daysbefore induction of peritonitis. This supplementation was prepared inmicro-emulsion containing: 0.3% ascorbyl palmitate, 0.3% alphatocopherol, 9.34% medium chain triglycerides 13% polysorbat 80. Themicro-emulsion without any nutrients served as placebo. The animalsdrank 4 ml/water per day; thus the phytonutrient intake of Lyc-O-Mato®,lutein, carnosic acid was 10:4.9:6.6 mg/Kg/day, respectively. Sterileperitonitis was induced by intraperitoneal injection of a sterilethioglycollate solution (4% w/v in PBS). In this model neutrophils arerecruited to the peritoneal cavity (the site of inflammation) during thefirst 24 h and than replaced by monocyte-macrophages. Peritoneal cellswere harvested after 24 h to isolate neutrophils or after 4 days tocollect macrophages using two washes of the peritoneal cavity with 8 mlRPMI medium. Peritoneal cells were washed three times with PBS and, whenappropriate, hypotonic lysis of erythrocytes was performed, yielding ahigh homogenous (90%) neutrophil cell population harvested 24 h afterthe induction of peritonitis and a high homogenous (90%) macrophage cellpopulation collected 4 days later. Peritoneal cells were identified byflow microfluorimetry on FACS (Becton Dickinson, Mountain View, Calif.)using FITC-conjugated rat anti-mouse neutrophils (MCA771F),FITC-conjugated rat anti-mouse F4/80 (MCA497F), and FITC-conjugated ratanti-mouse CD3 (MCA500F) (Serotec, Oxford, England) for thecharacterization of neutrophils, monocyte-macrophages and lymphocytes,respectively.

Body Weight

The body weights of each mouse was taken on the first day of thenutrient or placebo supply and at termination. There was no differencein weight gain between the groups studied.

Superoxides Production by Neutrophils

The production of superoxide anion (O₂ ⁻) by neutrophils was measured asthe superoxide dismutase-inhibitable reduction of ferricytochrome c bythe microtiter plate technique. Neutrophils (5×10⁵ cells/well) suspendedin 100 μl HBSS containing ferricytochrome c (150 mM). Stimulation wasinduced with PMA (50 ng/ml). The reduction of ferricytochrome c wasfollowed by a change of absorbance at 550 nm at 2 min intervals for 30min on a Thermomax Microplate Reader (Molecular Devices, Melno Park,Calif., USA). The maximal rates of superoxide generation were determinedand expressed as nanomoles O₂ ⁻/10⁶ cells/10 min using the extinctioncoefficient E₅₅₀=21 mM⁻¹ cm⁻¹.

Macrophage Cell Culture

Peritoneal macrophages were cultured in RPMI 1640 medium containing 10%FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin(Beit-Haemek, Israel) in 96-well plates (2× 10⁵ cells/well). LPS wasadded and the macrophages were cultured at 37° C. in 5% CO₂ atmospherefor 16-24 h.

NO Production Assay

NO levels in supernatants of cell cultures were determined by assayingnitrite levels using Griess reagent and sodium nitrite as a standard.

PGE₂ Production Assay

Supernatants of cell cultures were collected and immediately stored at−70° C. PGE₂ levels were determined by utilizing dextran coated charcoalradioimmunoassay protocol. Briefly, 100 μl sample or PGE₂ standard(Sigma Israel, Rehovot, Israel) were incubated in the presence of 500 μlanti-PGE₂ anti-serum (Sigma Israel, Rehovot, Israel) for 30 min.[³H]PGE₂ (Amersham Biosciences, NJ, USA) was added next for 24 h at 4°C. 24 h later, 200 μl cold dextran coated charcoal suspension was addedto each tube and incubated for 10 min on ice. The tubes were centrifugedat 3500 RPM for 15 min at 4° C. 500 μl of supernatants containing[³H]PGE₂-anti-PGE₂ complexes were counted (Packard Spectrometry 1900CA)and the amount of PGE₂ was calculated.

TNFα Production Assay

Concentrations of TNFα in supernatants of cell cultures, that werecollected and immediately stored at −70° C., were quantified by ELISAkits (Biolegend Inc., San Diego, Calif.).

Statistical Analysis

Data are presented as the mean±SEM. Statistical significance forcomparisons between groups was determined using Student's pairedtwo-tailed t-test.

Results

Superoxide production was measured in mice peritoneal neutrophilsisolated at 24 h of peritonitis induction (n=10 mice in each group).FIG. 6 presents superoxide production by non stimulated peritonealcells. These cells are primed due to the thioglicollate peritonealinjection and reflect the situation of the cells in a site ofinflammation. As shown there is a significant (p<0.01) reduction (39%)in the release of superoxide by the nutrient mixture treatment comparedwith the placebo (3.82±1.52 compared with 6.22±1.53 nmoles O₂/10⁶cells/min, respectively).

Moreover, stimulation of the cells (as in case of infections) resultedwith an effective release of superoxides (7.5 fold higher than withoutstimulation) and with no significant differences (p=n.s.) in superoxiderelease by cells of placebo mice (28.96 and 34.84 nmoles O₂/10⁶cells/min, respectively). The rate of stimulates superoxide productionof cells from the supplementation (mix) treated mice is sufficient tocombat infection.

These results demonstrate the significant anti inflammatory effect ofthe combination of LycoMato, Astaxanthin and Carnosic acid mixture thatsignificantly reduced the release of spontaneous free radicals byneutrophils arriving to the site of inflammation. Thus the nutrientfeeding prevented the delirious effect of neutrophils in the site ofinflammation but did not diminish their potential to release superoxidewhen attacked by infection.

The anti-inflammatory effect of the nutrient treatment was studied alsoon pro-inflammatory agents released by macrophages at the site ofinflammation. Mice received the supplementation or placebo for 7 daysbefore induction of peritonitis for 4 days (n=10 mice for each group).Supernatant of isolated peritoneal cells cultured for 24 h with 0.5ug/ml LPS was analyzed for NO production, PGE₂ production and TNFαproduction.

As shown in FIG. 7, there is a reduction (significance p<0.05) in NOproduction by peritoneal cells harvested from mice undergoing nutrientdrinking compared with placebo; 2.35±0.36 compared with 3.72±0.88 μM,respectively. Similarly, there is a reduction (significant p<0.05) inPGE₂ production by peritoneal cells harvested from mice undergoingnutrient drinking compared with placebo, 1.75±0.26 compared with2.72±0.33 ng/ml. Like wise TNFα production by peritoneal cells harvestedfrom mice undergoing nutrient drinking compared with placebo, 78.91±13.8compared with 99.55±24.03 pg/ml.

The invention claimed is:
 1. A composition comprising astaxanthin andtomato lycopene, wherein the molar concentration ratio of astaxanthin totomato lycopene is from 1:2 to 2:1.
 2. The composition of claim 1,wherein said composition is an oral composition.
 3. The composition ofclaim 1, further comprising a nutraceutical or a pharmaceuticalacceptable excipient.
 4. The composition of claim 1, wherein saidcomposition has a synergistic anti-inflammatory effect.
 5. A method oftreating a subject afflicted with inflammation, comprising the step ofadministering to said subject a therapeutically effective amount of acomposition comprising astaxanthin, and tomato lycopene in a molarconcentration ratio of said astaxanthin to said tomato lycopene from 1:2to 2:1, thereby treating a subject afflicted with inflammation.
 6. Themethod of claim 5, wherein said treating a subject afflicted withinflammation is inhibiting the production of a pro-inflammatory mediatorin a macrophage at a site of inflammation in said subject.
 7. The methodof claim 5, wherein said treating a subject afflicted with inflammationis inhibiting the production of NO, PGE2, TNF-alpha, or any combinationthereof in said subject.
 8. The method of claim 5, wherein said treatinga subject afflicted with inflammation is inhibiting the recruitment ofneutrophils to the site of inflammation, inhibiting neutrophilsactivation at the site of inflammation, or a combination thereof in saidsubject.
 9. The method of claim 5, wherein the composition is an oralcomposition.
 10. The method of claim 5, wherein the composition furthercomprises a nutraceutical or a pharmaceutical acceptable excipient.